The present invention relates generally to compositions and methods useful for inhibiting the multiplication of human immunodeficiency virus-1 (HIV-1) in infected patients, symptomatic or asymptomatic, and for attenuating HIV-1 multiplication following primary infection in previously uninfected subjects, thus minimizing progression to AIDS.
A variety of approaches to the treatment of human immunodeficiency virus type 1 (HIV-1) have focused on the transactivating (tat) gene of HIV-1, which produces a protein (Tat) essential for transcription of the virus. The tat gene and its protein have been sequenced and examined for involvement in proposed treatments of HIV (see, e.g., U.S. Pat. Nos. 5,158,877; 5,238,882; and 5,110,802; International Patent Application Nos. WO92/07871, WO91/10453, WO91/09958, and WO87/02989, published May 14, 1992, Jul. 25, 1991, Jul. 11, 1991 and May 21, 1987, respectively). Tat protein is released extracellularly, making it available to be taken up by other infected cells to enhance transcription of HIV-1 in the cells and by noninfected cells, to alter host cell gene activations. Tat renders the cells susceptible to infection by the virus. Uptake of Tat by cells is very strong, and has been reported as mediated by a short basic sequence of the protein (S. Fawell et al., Proc. Natl. Acad. Sci., USA, 91:664-668 (1994)).
Immunization with HIV-1 Tat protein as a potential AIDS vaccine is under active investigation. The HXB/LAV HIV-1 Tat sequence has been used as the immunogen in reported studies, either as a recombinant protein (A. Cafaro et al, Nat. Med., 5:643-650 (1999)), a DNA vaccine (S. Calarota et al, Lancet, 351:1320-5 (1998)), inactivated protein (Tat toxoid) (S. S. Cohen et al, Proc. Natl. Acad. Sci. USA, 96(19):10842-10847 (1999); A. Gringeri et al, J. Hum. Virol., 1:293-8 (1998)) or a DNA vaccine expressing inactive Tat (E. Caselli et al, J. Immunol., 162:5631-5638 (1999)). Immunizations with the full Tat sequence induced both cellular and humoral immunity. See, also, M. C. Rhe et al, J. Acquir. Immmune Defic. Syndr. Hum. Virol. 10:408-416 (1995); C. J. Li et al, Proc. Natl. Acad. Sci. USA, 94:8116-8120 (1997); and others).
International Patent Application No. WO92/14755, published Sep. 3, 1992, relates to the Tat protein and to the integrin cell surface receptor capable of binding to the Tat protein. Two Tat sequences that bind integrin are identified: -Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- (SEQ ID NO: 1), as well as -Gly-Arg-Gly-Asp-Ser-Pro- (SEQ ID NO: 2). These sequences are the basic region or domain which is the dominant binding site for the integrin. This specification demonstrates that a number of peptides corresponding to these Tat sequences and the corresponding integrins block in vitro cell binding to Tat coated plates, as do antibodies to the appropriate integrins. However, the specification also shows that these reagents do not block uptake of functional Tat by cells (see Example 9 in WO92/14755), thus nullifying the proposed mechanism of action for therapeutic benefit in HIV infection. The Tat sequences described in this international application are distinct from the peptide immunogens of the present invention.
Both monoclonal and polyclonal antibodies to Tat protein have been readily produced in animals and shown to block uptake of Tat protein in vitro (see, e.g., D. Brake et al, J. Virol., 64:962 (1990); D. Mann et al, EMBO J., 10:1733 (1991); J. Abraham et al, cited above; P. Auron et al, cited above; M. Jaye et al, cited above; G. Zauli et al, cited above). More recent reports showed that monoclonal or polyclonal antibodies to Tat protein added to tissue culture medium attenuated HIV-1 infection in vitro (L. Steinaa et al, Arch. Virol., 139:263 (1994); M. Re et al, cited above; and G. Zauli et al, J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 10:306 (1995)).
The inventor""s own publications (G. Goldstein, Nature Med., 2:960 (1996); and International Patent Application No. WO95/3 1999, published Nov. 30, 1995) reviewed the evidence indicating that secretion of HIV-1 Tat protein from infected cells and uptake by both infected and uninfected cells was important for the infectivity of HIV-1. Previous studies also showed that antibodies to Tat protein in vitro blocked uptake of Tat and inhibited m vitro infectivity. Active immunization of mammals was suggested to induce antibodies to HIV-1 Tat protein as a potential AIDS vaccine. See, also, G. Goldstein et al, xe2x80x9cMinimization of chronic plasma viremia in rhesus macaques immunized with synthetic HIV-1 Tat peptides and infected with a chimeric simian/human immunodeficiency virus (SHIV33)xe2x80x9d, Vaccine, 18:2789 (2000).
Other publications by the inventor, International Patent Application No. WO99/02185, published Jan. 21, 1999, and U.S. Pat. No. 5,891,994, issued Apr. 6, 1999 (both incorporated by reference herein), revealed a new concept in treatment and prevention of HIV-1 infection that utilized Tat sequences which were recognized as epitopes by the rabbit immune system. Unlike the prior disclosures discussed above, these publications relate to therapeutic and immunogenic combinations requiring at least two, and preferably all four, of the Tat peptides or polypeptides comprising the xe2x80x9cEpitope Ixe2x80x9d sequences spanning Tat amino acid residues 4 (or 5) through 10, as follows: -Asp-Pro-X7-Leu-Glu-Pro-(SEQ ID NO: 3) or R1-Val-Asp-Pro-X7-Leu-Glu-Pro-R2 (SEQ ID NO: 4), wherein X7 iS Arg, Lys, Ser or Asn. Such compositions induce antibodies that react with most HIV-1 Tat proteins and impair the multiplication of HIV-1. According to this publication, certain other Tat sequences, which comprise an xe2x80x9cEpitope IIxe2x80x9d peptide or polypeptide spanning Tat amino acid residues 41-50 of the formula R3-Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-R4 (SEQ ID NO:5), wherein X42 is selected from the group consisting of Gly or Ala, may be added to this composition. Alternatively, an xe2x80x9cEpitope IIIxe2x80x9d peptide or polypeptide spanning Tat amino acid residues 56-62 of the formula R5-Arg-Arg-X58-Z59-A60-Y61-Ser-R6 (SEQ ID NO:6), wherein X58 is selected from the group consisting of Ala, Pro, Ser and Gln; wherein Y61 is selected from the group consisting of Asp, Asn, Gly and Ser; wherein Z59 is selected from the group consisting of Pro and His; wherein A60 is selected from the group consisting of Gln and Pro, may be added to this composition. Still alternatively, an xe2x80x9cEpitope IVxe2x80x9d peptide or polypeptide spanning Tat AA residues 62-73 of the formula R7-Ser-Gln-X64-His-Gln-Y67-Ser-Leu-Ser-Lys-Gln-Pro-R8 (SEQ ID NO:7), wherein X64 is selected from the group consisting of Asn and Thr; wherein Y67 is selected from the group consisting of Ala and Val, may be added to this composition. The composition itself may be employed to induce antibodies to a large number of Tat sequences characteristic of the multiple variants of HIV-1. The compositions or antibodies generated are used as vaccine or prophylactic treatments against these multiple variants.
Despite the growing knowledge about HIV-1 disease progression, there remains a need in the art for the development of compositions and methods for treatment of HIV-1, both prophylactically and therapeutically, which are useful to lower the viral levels of HIV-1 for the treatment and possible prevention of the subsequent, generally fatal, AIDS disease.
In one aspect, the present invention provides a composition comprising at least two variants of a peptide or polypeptide of the Epitope I formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 (SEQ ID NO:8), wherein Y7 is selected from the group consisting of Arg, Lys, Ser and Asn; wherein X9 is selected from the group consisting of Glu and Asp; wherein Z12 is selected from the group consisting of Lys and Asn; wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl; wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. In this composition, at least one of the two variants must have the formula wherein Y7 is Arg and Z12 is Lys, and at least a second of the two variants must have the formula in which Y7 is Asn and Z12 is Asn. Each peptide of this composition is recognized as an HIV-1 Tat Epitope I by a primate immune system. This formula permits the construction and use of a variety of peptide combinations.
In another aspect, the above-described composition further contains one or more additional peptide or polypeptide(s) which represent other amino acid sequences which correspond to HIV-1 Tat amino acid residues 5 through amino acid residue 12. These optional amino acid sequences are described in detail below. These sequences are preferably from an HIV-1 strain with a Tat protein variant at that location.
In another aspect, this invention provides a composition described above that contains peptides or polypeptides which comprise at least the two required Epitope I peptides, recognized by primates (and preferably additional Epitope I peptides), in combination with one or more HIV-1 Tat Epitopes II, III and/or IV. Epitopes II, III and IV are the HIV-1 Tat peptide formulae described in International Patent Publication No. WO99/02185, incorporated herein by reference. Such compositions can combine appropriate HIV-1 Tat peptides, so as to provide for a composition that induces antibodies reactive with greater than about 95% of all known HIV-1 Tat proteins.
In yet a further aspect, the invention provides an antibody composition comprising at least one antibody, preferably generated in a primate, which specifically binds to a peptide or polypeptide of the formula R1-Asp-Pro-Y7-Leu-X9 -Pro-Trp-Z12-R2 (SEQ ID NO:8), wherein Y7 is selected from the group consisting of Arg, Lys, Ser and Asn; wherein X9 is selected from the group consisting of Glu and Asp; wherein Z12 is selected from the group consisting of Lys and Asn; wherein R1 is selected from the group consisting of hydrogen, a lower alkyl, a lower alkanoyl, and a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl; wherein R2 is selected from the group consisting of a free hydroxyl, an amide, and a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. This antibody composition preferably comprises at least two antibodies, i.e., one antibody which binds to the Epitope I variant in which Y7 is Arg and Z12 is Lys, and at least a second antibody which binds to a second Epitope I variant in which Y7 is Asn and Z12 is Asn. Other antibodies directed to other variants than the two specified variants may also be included in this composition. These antibodies in the composition bind to Epitope I sequences recognized by the primate immune system, which epitope is present on multiple variants of HIV-1 Tat proteins. These antibodies include a variety of antibody constructs, such as monoclonal antibodies, as described in detail below.
In still another aspect, the invention provides an antibody, particularly a monoclonal antibody, which specifically binds to a primate-recognized epitope of an HIV Tat protein, the epitope comprising the amino acid sequence -Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-(SEQ ID NO:9), wherein Y7, X9 and Z12 are defined as above.
In yet another aspect, the invention provides an antibody composition comprising at least one antibody that recognizes Epitope II peptide sequence -Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-(SEQ ID NO: 10), where X42 is Gly or Ala, as a distinct epitope from previously described antibodies which recognize the epitope of -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-(SEQ ID NO: 11). Preferably, the composition comprises one antibody which recognizes both the peptide in which X42 is Gly and the peptide in which X42 is Ala. These antibodies are preferably generated in primates. These antibodies in the composition bind to Epitope II sequences recognized by the primate immune system, which epitope is present on multiple variants of HIV-1 Tat proteins. These antibodies include a variety of antibody constructs, as described in detail below.
In still another aspect, the invention provides an antibody, preferably a monoclonal antibody, that recognizes Epitope II peptide sequence -Lys-X42-Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-(SEQ ID NO: 10), where X42 is Gly or Ala, as a distinct epitope from the epitope of -Leu-Gly-Ile-Ser-Tyr-Gly-Arg-Lys-(SEQ ID NO: 11), recognized by previously described antibodies.
In yet a further aspect, the invention provides a recombinant or synthetic gene which encodes sequentially a peptide or polypeptide that contains at least two variants of a peptide or polypeptide of the Epitope I formula R1-Asp-Pro-Y7-Leu-X9-Pro-Trp-Z12-R2 (SEQ ID NO: 8), as defined above. In this synthetic gene, at least one of the two variants must have the formula wherein Y7 is Arg and Z12 is Lys, and at least a second of the two variants must have the formula in which Y7 is Asn and Z12 is Asn. Optionally, this synthetic gene comprises a carboxy terminal Epitope II peptide, as recognized by the primate immune system. Alternatively, the recombinant or synthetic gene contains the seven or eight preferred primate-recognized Epitope I amino acid sequences identified below. The synthetic gene may contain each amino acid sequence separated by a spacer sequence, or may express each peptide/polypeptide in an open reading frame with a carrier protein. The synthetic gene may be separated from the carrier protein by a spacer if the spacer is fused to a primate-recognized Epitope I sequence, leaving an Epitope II sequence at the carboxy terminus of the recombinant protein. Further embodiments include multiple Epitope I peptides of the above formula fused together and to the carrier protein.
In yet a further aspect, the invention provides a synthetic molecule, e.g., a vector, comprising the above-described synthetic gene, operatively linked to regulatory nucleic acid sequences which direct and control expression of the product of the synthetic gene in a host cell.
In another aspect, the invention provides a recombinant microorganism, e.g., a virus or commensal bacterium, which contains the above described synthetic gene or synthetic molecule. This microorganism is capable of expressing multiple copies of the product of the gene or molecule in a host.
Still another aspect of the invention is a pharmaceutical composition useful for inducing antibodies that react with a large number of known HIV-1 Tat proteins, e.g., greater than 95%, and preferably greater than 99%, of the known Tat proteins. These induced antibodies can impair the multiplication of HIV-1. The pharmaceutical composition comprises at least one of the recombinant or synthetic peptide/polypeptide compositions described above; or the synthetic gene/molecule described above; or the recombinant microorganism described above, in a pharmaceutically acceptable carrier.
Still a further aspect of the invention is a pharmaceutical composition useful for impairing the multiplication of HIV-1, this composition containing an above described antibody composition or monoclonal antibody composition.
In yet a further aspect of the invention, a method for reducing the viral levels of HIV-1 involves exposing a human or other primate to antibody-inducing pharmaceutical compositions described above, actively inducing antibodies that react with most HIV-1 Tat proteins, and impairing the multiplication of the virus in vivo. This method is appropriate for an HIV-1 infected subject with a competent immune system, or an uninfected or chronically infected, but asymptomatic, subject. The method induces antibodies which react with HIV-1 Tat proteins, and which reduce viral multiplication during any initial acute infection with HIV-1 and which further minimize chronic viremia which leads to AIDS.
In still another aspect, the invention provides a method for reducing the viral levels of HIV-1 by administering to a human, who is incapable of mounting an effective or rapid immune response to infection with HIV-1, a pharmaceutical composition containing the antibody compositions described above. The method can involve chronically administering the composition.
Yet other aspects of the invention include methods for producing the compositions described above, as well as host cells transfected with such compositions.
Still another aspect of this invention is a kit useful for the measurement and detection of titers and specificities of antibodies induced by immunization with the compositions described above. The kit of the invention includes preferably the two required Epitope I peptides described above, as well as addition peptides of the Epitope I, recognized by primates, and possibly additional peptides of Epitopes II through IV, and coated solid supports, a labeled reagent for detecting the binding of antibodies to these peptides, and miscellaneous substrates and apparatus for evoking or detecting the signals provided by the labels, as well as conventional apparatus for taking blood samples, appropriate vials and other diagnostic assay components.
In yet a further aspect, the invention provides a method for detecting the titers and reactivity patterns of antibodies in subjects immunized with the compositions of this invention. The method includes the steps of incubating dilutions of the subject""s biological fluid, e.g. serum, with plates or beads on which are bound one or more peptides of the Epitope I sequences of this invention and optionally, the Epitopes II through IV, washing away unbound biological materials, and measuring any antibody binding to the peptides with labeled reagent, e.g., an anti-human immunoglobulin to which is associated an enzyme. Depending on the type of label employed, the signal produced by the label may be evoked by further adding a substrate which reacts with the enzyme, e.g., producing a color change. Other conventional labels may also be incorporated into this assay design.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.